Circulating cell-free miRNAs have emerged as promising minimally-invasive biomarkers for early detection, prognosis and monitoring of cancer. They can exist in the bloodstream incorporated into extracellular vesicles (EVs) and ribonucleoprotein complexes.
Trang 1R E S E A R C H A R T I C L E Open Access
Detection of circulating miRNAs:
comparative analysis of extracellular
vesicle-incorporated miRNAs and cell-free
miRNAs in whole plasma of prostate cancer
patients
Edgars Endzeli ņš1 †, Andreas Berger1†, Vita Melne1,2, Cristina Bajo-Santos1, Krist īne Soboļevska1
, Art ūrs Ābols1
, Marta Rodriguez3, Daiga Šantare4
, Anastasija Rud ņickiha1
, Vilnis Lietuvietis1,2, Alicia Llorente3and Aija Lin ē1*
Abstract
Background: Circulating cell-free miRNAs have emerged as promising minimally-invasive biomarkers for early detection, prognosis and monitoring of cancer They can exist in the bloodstream incorporated into extracellular vesicles (EVs) and ribonucleoprotein complexes However, it is still debated if EVs contain biologically meaningful amounts of miRNAs and may provide a better source of miRNA biomarkers than whole plasma The aim of this study was to systematically compare the diagnostic potential of prostate cancer-associated miRNAs in whole plasma and in plasma EVs
Methods: RNA was isolated from whole plasma and plasma EV samples from a well characterised cohort of 50 patient with prostate cancer (PC) and 22 patients with benign prostatic hyperplasia (BPH) Nine miRNAs known to have a diagnostic potential for PC in cell-free blood were quantified by RT-qPCR and the relative quantities were
Results: Only a small fraction of the total cell-free miRNA was recovered from the plasma EVs, however the
EV-incorporated and whole plasma cell-free miRNA profiles were clearly different Four of the miRNAs analysed showed a diagnostic potential in our patient cohort MiR-375 could differentiate between PC and BPH patients when analysed in the whole plasma, while miR-200c-3p and miR-21-5p performed better when analysed in plasma EVs EV-incorporated but not whole plasma Let-7a-5p level could distinguish PC patients with Gleason score≥ 8 vs ≤6
Conclusions: This study demonstrates that for some miRNA biomarkers EVs provide a more consistent source of RNA than whole plasma, while other miRNAs show better diagnostic performance when tested in the whole plasma
Keywords: Prostate cancer, Cell-free miRNAs, Extracellular vesicles, Exosomes, Microvesicles, Biomarkers, Liquid biopsy
* Correspondence: aija@biomed.lu.lv
†Equal contributors
1 Latvian Biomedical Research and Study Centre, Ratsupites Str 1, k-1, Riga
LV-1067, Latvia
Full list of author information is available at the end of the article
© The Author(s) 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver
Trang 2Circulating cell-free micro-RNAs (miRNAs) have emerged
as promising biomarkers for the development of
blood-based assays for early detection, prognosis and monitoring
of cancer In 2008, Mitchell et al demonstrated for the first
time that miRNAs are released from prostate cancer (PC)
cells into the bloodstream, where they exist in a remarkably
stable form [1] miRNAs were shown to remain stable after
incubation of plasma or serum at room temperature for up
to 24 h and to resist RNase A digestion, HCl and NaOH
treatment or multiple freeze-thaw cycles [1, 2]
Subse-quently, the levels of circulating miRNAs have been studied
in patients with various cancers, including PC, resulting in
the discovery of individual miRNAs or miRNA signatures
with diagnostic and/or prognostic value [3]
PC is the most frequently diagnosed cancer in males in
Europe and the United States [4, 5] Currently, the serum
PSA test is the most commonly used tool for organised
screening programs, opportunistic screening and
monitor-ing of PC [6] However, PSA is not cancer specific and the
high false-positive rate and low specificity leads to large
numbers of unnecessary prostate biopsies and emotional
morbidity [7] Furthermore, PC is characterised by a highly
heterogeneous course - one part of the patients develops a
high-grade disease with extracapsular spread and distant
metastases requiring aggressive treatment, while others
have a relatively indolent, slowly progressing disease that
could have been managed by active surveillance [8] The
current standard of care analyses, however, do not predict
whether a histologically proven tumour will give rise to a
clinically significant disease, leading to overtreatment of
indolent PC Hence, the greatest unmet clinical needs in
the management of PC are sensitive and reliable
non-invasive tools for differentiating between PC and benign
prostatic diseases, and between potentially fast progressing
PC requiring aggressive treatment and a relatively indolent
disease that can be managed by active surveillance
More than 20 studies investigating levels of cell-free
miRNAs in plasma or serum of PC patients have been
published up to date [9, 10] The majority of these studies
were focused on the identification of circulating miRNAs
that differentiate between patients with PC and benign
prostatic hyperplasia (BPH) or healthy controls Some of
these studies have shown remarkably high diagnostic
value For example, Chen et al identified a 5 miRNA panel
that could differentiate PC from BPH with an AUC of
0.924 and PC from healthy controls with an AUC of 0.860
[11] Some other studies have reported cell-free miRNAs
that differentiate between localised and metastatic
castra-tion resistant prostate cancer (mCRPC) or between
low-grade and high-low-grade PC For example, Mihelich et al
developed a“miRScore” that based on the serum levels of
14 miRNAs could predict absence of high-grade PC
among men with PC and BPH with a negative predictive
value of 0.939 [12] However, relatively few miRNA biomarkers have been validated by several independent studies, while many other miRNAs either have been reported in a single study or show conflicting results [3, 10] Therefore, the analysis of cell-free miRNAs is regarded
as a poorly reproducible technique [3, 13, 14]
Cell-free miRNAs circulating in the bloodstream have been found to be enclosed into extracellular vesicles (EVs) [15, 16], or to exist in a vesicle-free form associated with high-density lipoproteins [17], Ago2 protein [18, 19] or other RNA binding proteins [20] The majority of the studies has used whole plasma or serum as a source of cell-free miRNAs However, it has recently been hypothe-sised that cancer-derived EVs may be enriched with miRNA signatures reminiscent of their cell of origin, con-tain rare yet highly specific RNA biomarkers and protect their RNA cargo from degradation in the bloodstream and, therefore, the analysis of EV-enclosed miRNAs may be su-perior to whole plasma/serum analysis [10, 21, 22] Never-theless, to the best of our knowledge, a direct comparison
of miRNA detection assays in whole plasma and plasma EVs has not been reported so far
In this study, we evaluated the performance of 9 miRNA biomarkers previously reported to have a diagnostic or prognostic significance in PC by quantifying them in the whole plasma and plasma EVs in a cohort of 50 PC and
22 BPH patients
Methods
Study population and sample collection
Patients with PC and BPH were recruited between September 2011 and December 2013 at Riga East University Hospital and subsequently were followed up until December 2016 The diagnosis was established using standard of care diagnostic examinations and Gleason score was determined according to standard histopathological criteria by an experienced pathologist Pre-treatment blood samples were collected into EDTA-coated tubes and processed at room temperature within
2 h of blood draw Plasma samples were centrifuged twice for 10 min at 2000 g, aliquoted and stored at−80 °C until analysis The samples were deposited into the Latvian Genome Database Biobanking procedures were approved
by the Committee of Medical Ethics of Latvia and the use
of clinical samples for the research was approved by the Committee of Biomedical Ethics of Riga East University Hospital The blood samples were collected after the patients’ informed written consent was obtained
The following groups of patients were selected from the Database: PC with Gleason score ≥ 8 (Gleason high,
n = 24), PC with Gleason score ≤6 (Gleason low, n = 26) and BPH (absence of PC confirmed by histological examination of ultrasound-guided needle biopsies and
no change in the diagnosis within the follow-up period,
Trang 3n = 22) Clinical data of the study population are
provided in Table 1 In addition, plasma samples from 5
PC patients and 5 healthy controls were used for the
quality control of EV isolation
Isolation of extracellular vesicles
EVs were isolated from 400 μl of plasma using size
exclusion chromatography (SEC) SEC columns were
pre-pared by filling TELOS SPE columns (Kinesis, USA) with
10 ml (bed volume) of CL6B sepharose (GE Healthcare,
USA) Plasma samples were loaded on the columns and
gravity-eluted with PBS The eluate was collected in 12
sequential 0.5 ml fractions Each fraction was measured by
Zetasizer Nano ZS (Malvern, UK) and fractions
contain-ing particles larger than 30 nm were combined and
con-centrated to 100μl using Amicon Ultra 3 kDa centrifugal
filters (Merck, Millipore, Germany)
Transmission electron microscopy
Ten μl of EV suspension in PBS were applied to
300-mesh carbon coated copper EM grid and incubated for
5 min Then the samples were negatively stained with 1% uranyl formate (w/v) for 1 min, dried and examined using JEM-1230 transmission electron microscope (JEOL, USA)
Nanoparticle tracking analysis
Size distribution profile and concentration of EVs was de-termined using NanoSight NS500 instrument (Malvern, UK) EV samples were diluted 1000–25,000 fold in PBS to achieve particle concentration in range from 1×108 to 1×109particles/ml For each sample, five 30 s videos were recorded with the following settings: 25C, 0.944–0.948 cP,
1259 slider shutter, 366 slider gain, and 11 camera level The data analysis was performed with NanoSight NTA Software v3.1 Build 3.1.54 in the auto mode
Western blot
EVs and PC-3 cells (used as a positive control) were lysed in RIPA buffer (150 ml NaCl, 1% Triton X-100, 0.5% Na deoxycholate, 0.1% SDS, 50 ml Tris) and the protein concentration was assessed using Pierce™ BCA
Table 1 Clinical characteristics of the study population
Age (years)
Serum PSA (ng/ml)
Gleason score
Metastasis status
Cancer grade
Prostatitis
Trang 4Protein Assay Kit (Thermo Fisher Scientific, USA)
fol-lowing manufacturer’s instructions Thirty micrograms
of EV and cell proteins were mixed with Laemmli buffer
under reduction conditions, denatured for 5 min at 100 °C
and loaded on 10% SDS-PAGE gel Proteins were
electro-blotted to nitrocellulose membranes and the membranes
were blocked with 10% (w/v) fat-free milk and then
incu-bated with the following primary antibodies: anti-TSG101
(Abcam, # ab125011), Calnexin (Abcam, # ab22595), CD9
(Santa Cruz Biotechnology, # sc-13118) and β-actin
(Abcam, # ab8224) in 1:1000 dilution The blots were
washed and incubated with horseradish
peroxidase-conjugated goat anti-rabbit IgG F(ab’)2-HRP (1:2000)
(Santa Cruz, #sc-3837) or chicken anti-mouse IgG-HRP
(1:2000) (Santa Cruz, #sc-2962) secondary antibodies,
re-spectively Protein expression was visualized using
Western Blotting Detection Reagent kit (GE HealthCare
Lifesciences, Germany)
Enzymatic treatment
Prior to RNA extraction, EVs samples were treated with
1 mg/ml proteinase K (Thermo Fisher Scientific, USA) for
30 min at 37 °C Proteinase K was inactivated by
incubat-ing the samples for 10 min at 65 °C Then the samples
were treated with 10 ng/μl RNase A (Thermo Fisher
Scientific, USA) for 15 min at 37 °C
RNA extraction
RNA was extracted from EV and whole plasma samples
using miRNeasy Micro Kit (Qiagen, USA) according to
the manufacturer’s instructions with slight modifications
of the protocol Briefly, 5 volumes of QIAzol Lysis
Reagent were added to each sample Subsequently,
sam-ples were spiked with 1μl of UniSp6 (Exiqon, Denmark),
which was used as a normaliser in downstream analysis
After adding 1 volume of chloroform samples were
cen-trifuged for 15 min at 12000 g at 4 °C and the aqueous
phase was transferred to a new tube Then, 1.5 volumes
of 100% ethanol were added to each sample and the
mixture was loaded onto a MinElute spin column
Columns were centrifuged at 1000 g for 30 s at room
temperature in each round until entire sample was
loaded RNA was eluted in 15 μl of RNase-free water
using low-bind tubes The quantity and quality of RNA
was assessed using Agilent 2100 Bioanalyzer and RNA
6000 Pico Kit (Agilent technologies, # 5067–1513)
RT-qPCR analysis
One third of each RNA sample isolated from EVs and
whole plasma was reverse-transcribed using miRCURY
LNA Universal cDNA Synthesis kit II (Exiqon)
accord-ing to the manufacturer’s protocol cDNA reaction
mix-tures were diluted 1:40 and 4 μl were used for qPCR
reactions qPCR was carried out using microRNA LNA
PCR primer sets and ExiLENT SYBR Green master mix (Exiqon) according to the manufacturer’s protocol on ViiA
7 Real-Time PCR system (Thermo Fisher Scientific)
Statistical analysis
Ct values were averaged between duplicates and normalized against UniSp6 spike-ins by subtracting them from average spike-in Ct values in the same samples, resulting in log2 relative quantities (log2 RQ’s) The statistical analyses were performed with GraphPadPrism 5 (GraphPad, USA) A non-parametric Mann-Whitney U test was used to com-pare the RQ values of each miRNA between the groups of samples Multiple testing correction was done by false discovery rate (FDR) estimation and adjusted (adj.) P-value
of ≤0.05 was considered to be significant To assess the diagnostic potential, the area under the ROC curve (AUC) was calculated for each miRNA
Results
Selection of miRNA biomarkers
Nine miRNAs, whose levels in plasma or serum have been reported to have a diagnostic or prognostic significance in
PC in at least two independent studies, were selected for this study Studies showing their relevance for the diagnosis
or prognosis of PC are summarised in Table 2 MiR-21-5p, miR-200c-3p, miR-210-3p and miR-375 have been shown
to be increased in the blood of PC patients as compared to BPH or healthy controls consistently by two or more stud-ies, while miR-30c-5p and miR-223-3p were found to be consistently decreased in the blood of PC patients Incon-sistent findings have been reported for Let-7a-5p, miR-141-3p and miR-106a-5p
Yield and purity of EVs
In order to compare the levels of the selected miRNAs in plasma EVs and whole plasma, each plasma sample was divided into two 400 μl aliquots – one was used for the isolation of EV-incorporated RNA, while another was used directly for the isolation of cell-free RNA from whole plasma according to the workflow shown in Fig 1a
To assess the yield and purity of EVs, EV samples from
5 PC patients and 5 healthy controls (not included in the miRNA analysis) were characterised by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blot analysis TEM images revealed that the majority of particles were ranging in size from 25 to 60 nm that corresponds to the size of exosomes (Fig 1b) However, as it has been shown that SEC-based EV isolation methods do not result in lipoprotein-free EV preparations [23], it cannot be excluded that a fraction of the particles are lipoproteins NTA showed that the concentrations of EVs range from 3.14×1010 to 1.27×1012 particles per ml of plasma (Fig 1c) The EV count was slightly increased in plasma
Trang 5Table 2 Circulating cell-free miRNA biomarkers for prostate cancer
miRNA Expression in PC tissues Level in blood
type
Patient groups and sample size Direction Normalisation Ref
Let-7a-5p
Down in PC vs adj.
Normal tissues
[ 45 ] Serum PC (n = 75), BPH (n = 27) Down in PC RNA input and
miR-16, miR-425
[ 52 ] Down in PC vs BPH [ 44 ] Serum High grade PC (n = 50),
low grade PC (n = 50), BPH (n = 50)
Down in high grade PC
vs low grade PC, BPH
RNA input and spike-ins
[ 12 ]
Serum Disseminated PC (n = 20),
BPH (n = 13)
Up in disseminated PC Spike-in and
miR-320a
[ 37 ]
miR-21-5p
Up in PC vs adj Normal
(n = 10)
[ 55 ] Plasma mCRPC (n = 25, pooled),
LPC (n = 25, pooled)
Similar in PC and adj.
Normal tissues (n = 36)
[ 56 ] Serum ADPC (n = 20), HRPC
(n = 10), LPC (n = 20), BPH (n = 6)
Up in HRPC vs ADPC, LPC U6 snRNA [ 42 ]
Up in PC vs
normal tissues
[ 57 ] Plasma PC (n = 51),
HC (n = 20)
miR-30c-5p
Up in PC vs adj Normal
epithelium (n = 37)
[ 58 ] Serum High grade PC (n = 50),
low grade PC (n = 50), BPH (n = 50)
Down in high grade PC
vs low grade, BPH
RNA input and spike-ins
[ 12 ]
Up in PC vs
normal tissues
[ 57 ] Plasma PC (n = 80), BPH (n = 44),
HC (n = 54)
Down in PC vs BPH, HC U6 snRNA [ 11 ] Serum PC (n = 36), HC (n = 12) Down in PC RNA input [ 51 ]
miR-106a-5p
Up in PC vs
normal tissues
[ 57 ] Serum High grade PC (n = 50),
low grade PC (n = 50), BPH (n = 50)
Down in high grade PC RNA input and
spike-ins
[ 12 ]
Serum PC (n = 36), HC (n = 12) Up in PC RNA input [ 51 ]
miR-141-3p
Up in mPC, PC vs
normal tissues
[ 53 ] Serum High grade PC (n = 50),
low grade PC (n = 50), BPH (n = 50)
Detectable in <50%
of patients
RNA input and spike-ins
[ 12 ]
Up in PC vs BPH [ 52 ] Serum PC (n = 75), BPH (n = 27) Up in PC RNA input and
miR-16, miR-425
[ 52 ]
Up in BCR after RP
vs no BCR after RP
[ 59 ] Plasma mCRPC (n = 25, pooled),
LPC (n = 25, pooled)
Serum mCRPC (n = 26), low-risk LPC
(n = 28)
Up in PC (n = 36)
vs normal tissue
(n = 36)
[ 54 ] Plasma EVs PC (n = 78), HC (n = 28) Up in PC Spike-ins [ 38 ] Serum
EVs
mPC (n = 47), non-recurrent
PC (n = 72)
Up in mPC
Serum 71 PC: N1 (n = 48), N0 (n = 23),
GS ≥8 (n = 29), GS = 7 (n = 42) Up in N1 PC vs N0 PC;Up in GS ≥ 8 vs GS = 7 Spike-ins [54] Plasma mPC (n = 25), LPC (n = 26) Up in mPC vs LPC; Similar
in PC and HC
RNU1A snRNA [ 43 ] Serum mPC (n = 25), HC (n = 25) Up in mPC Spike-ins [ 60 ] Serum PC (n = 54), non-malignant
(n = 79)
Up in higher GS; Similar in
PC and non-malignant
RNU1 –4 and SNORD43
[ 61 ]
miR-200c-3p
Up in PC vs
normal tissue
[ 62 ] Plasma mCRPC (n = 25, pooled),
LPC (n = 25, pooled)
Serum mCRPC (n = 25), HC (n = 25) Up in mCRCP Spike-ins [ 41 ]
miR-210-3p
Up in PC vs BPH [ 44 ] Serum PC (n = 31), BPH (n = 13) Up in PC Spike-in and
miR-320a
[ 37 ] Serum mCRPC (n = 21), HC (n = 20) Up in mCRCP Spike-ins [ 41 ]
miR-223-3p
Up in PC vs adj.
Normal tissues (n = 10)
vs low grade, BPH
RNA input and spike-ins
[ 12 ]
Trang 6from PC patients as compared to the healthy controls
(mean count in PC 7.08×1011 vs 4.15×1011 in healthy
controls), although the difference didn’t reach statistical
significance in our sample set The size distribution
analysis showed that the diameter for the majority of
particles was in the range from 50 to 150 nm with a
minor fraction reaching ~230 nm (Fig 1d), which is
somewhat inconsistent with the TEM results This
discrepancy likely has arisen due to the difference in the
minimum detectable EV size between both techniques
[24] and /or shrinking of EVs during fixation for TEM
[25] Western blot analysis showed that the EVs were
positive for typical EV markers TSG101 and CD9, and
negative for the endoplasmic reticulum protein Calnexin
(Fig 1e) Taken together, these results show that the EV
isolation method used in this study results in a relatively
high yield of exosome-enriched EV preparations without
detectable contamination of intracellular components
RNA profiles in EVs and whole plasma
As it has been suggested that EVs may associate with
lipoproteins or protein complexes that carry cell-free
miRNAs and protect them from degradation [18, 26], we
first tested the effect of proteinase K and RNase A
treat-ment on the miRNA levels in plasma EVs from three
healthy individuals (Fig 2a) Treatment of EVs with
RNase A alone reduced the relative quantity (RQ) values
by 15.5 to 43.6% for different miRNAs, while the
treatment with proteinase K prior to RNase A resulted
in the reduction of RQs by 50.4 to 69.3% This suggests that the proteinase K treatment is required for efficient removal of extra-vesicular RNA Therefore, in order to study the intraluminal miRNAs, all EV preparations were treated with proteinase K and RNase A prior to the RNA extraction RNA was extracted from EVs and whole plasma using miRNeasy Micro kit, which is designed for isolation of total RNA from small amounts
of sample Typical RNA profiles obtained by Bioanalyzer from whole plasma and EVs are shown in Fig 2b The profiles show the presence of small RNA peaks of 25 to
200 nt both in whole plasma and EVs, while 18S and 28S rRNA peaks are present in whole plasma and EVs without the enzymatic treatment (not shown) but not in the treated EVs, thus suggesting that the majority of rRNA is bound to the surface of EVs
Relative abundance of EV-incorporated miRNAs
An equal proportion (one third) from the total RNA amount obtained from the EV and whole plasma samples of PC and BPH patients was used for the RT-qPCR analysis of the 9 selected miRNA biomarkers Spike-ins were used to control for a variation in RNA extraction, cDNA synthesis and PCR efficiency and they typically varied less than by 1 Ct In order to assess the relative abundance of EV-enclosed miRNAs, a ratio between EV-enclosed and total cell-free miRNAs in
Table 2 Circulating cell-free miRNA biomarkers for prostate cancer (Continued)
miRNA Expression in PC tissues Level in blood
type
Patient groups and sample size Direction Normalisation Ref High grade PC (n = 50),
low grade PC (n = 50), BPH (n = 50)
Up in PC vs
normal tissues
[ 57 ] Serum PC (n = 36), HC (n = 12) Down in PC RNA input [ 51 ] miR-375 Up in mPC, PC
vs normal tissues
[ 53 ] Plasma EVs
CRPC (n = 100) High miRNA level
associated with poor OS
RNA input and miR-30a-5p, miR-30e-5p
[ 39 ]
Serum PC (n = 31), BPH (n = 13) Up in PC Spike-in and
miR-320a
[ 37 ]
Up in PC (n = 36) vs
normal tissue (n = 36)
[ 54 ] Plasma mCRPC (n = 25, pooled),
LPC (n = 25, pooled)
Serum mCRPC (n = 26), low-risk
LPC (n = 28)
Serum EVs
mPC after RP (n = 47), non-recurrent PC after
RP (n = 72)
Serum 71 PC: N1 (n = 48), N0
(n = 23), GS ≥8 (n = 29),
GS = 7 (n = 42)
Up in N1 PC vs N0 PC;
similar in GS ≥ 8 and GS = 7 Spike-ins [54]
ADPC androgen-dependent prostate cancer, BCR biochemical recurrence, BPH benign prostatic hyperplasia, CRPC castration resistant prostate cancer, EVs extracel-lular vesicles, HC healthy control, HRPC hormone-refractory prostate cancer, LPC localized prostate cancer, mCRPC metastatic castration resistant prostate cancer, mPC metastatic prostate cancer, PC prostate cancer, RP radical prostatectomy
Trang 7whole plasma was calculated (Fig 3a) The results
showed that only a small fraction of the cell-free miRNA
was retrieved from the EVs However, the EV-enclosed
fraction was not uniformly low – it varied from 6.36%
for Let-7a-5p to 0.65% for miR-210-3p Spearman
correlation analysis revealed only weak to moderate
correlation between EV-enclosed and whole plasma
cell-free miRNAs (Table 3) As an example, a paired dot plot
in Fig 3b shows the discordance in the Let-7a-5p levels
in EVs and whole plasma from the same patients These data support the idea that EV-enclosed miRNA profile dif-fers from cell-free miRNA profile in the whole plasma Clearly, the size of the EV-enclosed miRNA fraction depends on the efficacy of the EV isolation method and the obtained ratios are not expected to represent the EV-enclosed: EV-free miRNA ratio However, the NTA
Fig 2 Effects of proteinase K and RNase A treatment on the relative quantity of EV-incorporated miRNAs and RNA profiles in whole plasma and EVs a RT-qPCR analysis of miRNA levels in EVs treated with RNase A alone or with a combination of proteinase K and RNase A relatively to untreated EVs Bars show the mean percentage in EVs from 3 healthy individuals b A representative RNA profile from whole plasma and EVs treated with proteinase K and RNase A obtained by Bioanlyzer RNA 6000 Pico chip
Fig 1 Workflow of the study and characterisation of plasma EVs a Workflow of the study b Representative transmission electron microscopy image of plasma EVs c Quantification of EVs isolated from plasma of PC patients and healthy controls (HC) by nanoparticle tracking analysis d Average size distribution of EVs isolated from plasma of PC patients and healthy controls e Western blot analysis of EV markers (TSG101, CD9), endoplasmic reticulum protein Calnexin and β-actin in plasma EVs isolated from two healthy individuals and PC-3 cells (as a positive control)
Trang 8data showed that the EV count recovered in this study
was similar or even higher than that reported by other
studies [27–30], therefore we assume that the EV yield
in our study is representative of that obtained by the
current standard EV isolation techniques
These results show that although only a small fraction of
the total cell-free miRNA present in plasma was recovered
from EVs, the EV-incorporated miRNA profile is clearly
dif-ferent from that in the whole plasma
Diagnostic potential of EV-enclosed and total cell-free
miRNAs
To assess the diagnostic potential of the selected miRNAs,
their relative quantity in EVs and whole plasma was
com-pared between patients with PC (n = 50) and BPH
(n = 22) Three of the 9 miRNAs tested showed a
diagnos-tic value in our sample set (Fig 4) MiR-375 was
signifi-cantly increased in PC patients as compared to BPH (FDR
adj p = 0.03) and had an AUC of 0.68 (95% CI: 0.54–0.83,
p = 0.01), when tested in the whole plasma The same
ten-dency was observed for EV-enclosed miR-375, however it
didn’t reach statistical significance On the contrary,
miR-200c-3p and miR-21-5p could differentiate between PC and BPH better when tested in EVs than in the whole plasma (AUC of 0.68, p = 0.01 and 0.67, p = 0.02, respect-ively, when tested in EVs and AUC of 0.62, p = 0.12 and AUC of 0.61, p = 0.16, respectively, when tested in whole plasma) The levels of the other miRNAs were not signifi-cantly different in PC samples compared to BPH neither
in EVs nor in whole plasma
Next, we investigated the association of EV-enclosed and whole plasma miRNA levels with PC aggressiveness
We found that the level of Let-7a-5p was significantly decreased in EVs from PC patients with high Gleason score (≥8) compared to low Gleason score (≤6) and it could differentiate between these groups with AUC of 0.68 (95% CI: 0.52–0.84, p = 0.03) (Fig 5) Although the same tendency was observed in whole plasma, the stand-ard deviation was larger and statistical significance was not reached No other miRNA could differentiate between PC patients with high and low Gleason scores Finally, none of the miRNAs was associated with the presence of histologically confirmed prostatitis in PC and BPH patients, thus showing that the alterations in the miRNA levels are not due to prostatic inflammation
Discussion
Cells can release miRNAs to the extracellular space either incorporated into EVs [31, 32] or in a vesicle-free form bound to various protein and lipoprotein com-plexes [17–20] Quantification of these miRNAs in blood from cancer patients may offer new opportunities for diagnosis, prognosis, monitoring of treatment response and early detection of recurrence in a minimally invasive way However, human blood contains a complex mixture
of miRNAs derived from various cell types and, there-fore, robust quantification of cancer-derived cell-free miRNAs has turned out to be a challenging task [14] Currently, it is still debated if the EV-based miRNA
Fig 3 Relative abundance of EV-incorporated miRNAs a Ratio between EV-incorporated and total cell-free miRNAs in whole plasma Bars represent the mean ratios in groups of patients with PC and BPH b A paired dot plot shows the ranking of PC patients according to Let-7a-5p levels in EVs and whole plasma; lines connect the samples from the same individual
Table 3 Spearman correlation coefficients of EV-enclosed and
whole plasma miRNAs
miRNA Spearman r 95% confidence interval p value
Trang 9detection assays are superior to the whole plasma-based
assays miRNA profiles in cancer-derived EVs have been
found to be reminiscent of their cell-of-origin [31, 33],
though due to selective RNA sorting mechanisms they
may be enriched or depleted of some specific miRNAs
[34] The EV membrane protects the RNA cargo from
degradation in the bloodstream and the intraluminal
RNA content is thought to be relatively stable, therefore
EVs may provide a more consistent source of miRNA
biomarkers than whole plasma [15, 30] On the other
hand, it has been calculated that there is far less than
one molecule of a given miRNA per EV [35], which
raises the question of whether all EVs contain miRNAs
and if the amounts are biologically meaningful
Moreover, it can be argued that the EV isolation step may introduce a higher variation and result in a low RNA yield that in turn would lead to lower sensitivity, higher standard deviations and poor reproducibility of the EV-based miRNA assays as compared to whole plasma assays
Here, we have performed a systematic comparison of miRNA levels in whole plasma and EVs isolated from the same plasma samples in a well-characterised cohort of PC and BPH patients Our results show that EV-incorporated miRNA constitutes only a minor fraction of whole plasma miRNA This is in line with a study by Chevillet et al showing that exosome fractions contained a small minor-ity of the miRNA content of plasma [35] Nevertheless,
Fig 4 Circulating miRNA levels in patients with BPH and PC Scatter plots show the log2RQ values of each miRNA tested in EVs and in whole plasma FDR-adjusted p values are show at the top of each graph Area under the ROC curve (AUC), 95% confidence interval and p value for differentiating between PC and BPH is shown below each graph
Trang 10the miRNA levels in EVs and whole plasma were poorly
correlated, and the EV-incorporated and whole plasma
miRNA profile was clearly different This finding is
con-sistent with a NGS-based study by Cheng et al that
com-pared small RNA profiles in EVs, plasma and serum of 3
healthy individuals and showed that the miRNA levels
dif-fer remarkably between plasma and serum EVs and
between EVs and cell-free plasma and serum [30]
Three out of 9 miRNAs analysed could differentiate
be-tween PC and BPH patients in our cohort MiR-375
showed a better diagnostic performance when tested in
whole plasma as compared to EVs MiR-375 is an
onco-genic miRNA that is overexpressed in tumours with high
Gleason score and more advanced pathological stage [36]
Increased plasma or serum levels of miR-375 in patients
with PC vs BPH or metastatic CRPC vs localised PC have
been reported before in several studies (Table 2), and the
AUC obtained in our study was similar to that reported
be-fore [37] MiR-375 had one of the lowest EV to whole
plasma ratios among the miRNAs analysed in this study
and it was undetectable in a significant portion of EV
sam-ples It still may have diagnostic properties in cases where it
is detectable, though proving its diagnostic value would
re-quire a larger cohort of samples Two studies have reported
the presence of miR-375 in blood EVs from PC patients
Bryant et al showed that its level is increased in serum EVs
from patients with metastatic PC as compared to
non-recurring PC [38], and Huang et al reported that high
EV-miR-375 level is associated with a poor prognosis in CRPC
[39] Hence, increased levels of EV-incorporated miR-375
appear to be associated with metastatic disease As only 3
of the patients in our cohort had a metastatic disease at the
time of the blood draw, we reasonably detected it only in a
minority of PC patients in our cohort Moreover, as these
studies did not describe treatment of EVs with proteinase
K, it is possible that the EV preparations also contained
protein-bound miRNAs co-isolated with EVs
On the contrary, EV-incorporated 200c-3p and miR-21-5p showed better diagnostic performance than in whole plasma Increased plasma or serum levels of miR-200c-3p have been found before in patients with metastatic CRPC
as compared to localised PC or healthy controls [40, 41] Similarly, miR-21-5p has been reported to be increased in plasma or serum of patients with PC as compared to healthy controls and patients with CRPC as compared to localised PC [40, 42, 43] However, to the best of our know-ledge, an association of EV-incorporated miR-200c-3p and miR-21-5p with PC has not been reported before Hence, our study shows for the first time that EVs provide a better source for testing these miRNAs as PC biomarkers than whole plasma
The only miRNA biomarker that could differentiate between PC patients with high vs low Gleason score was EV-incorporated Let-7a-5p, whose level was decreased
in patients with Gleason score≥ 8 This is in line with a study by Mihelich et al showing that serum levels of Let-7a were decreased in PC patients with Gleason 4 + 5 grade tumours as compared with Gleason grade 3 [12] Our study, though, shows that the whole plasma and EV levels of Let-7a-5p are poorly correlated and that EV-incorporated Let7a-5p level is more informative than Let7a-5p in whole plasma
The cellular origin of circulating miRNAs is unclear Although it seems likely that oncogenic miRNAs such as miR-375, miR-200c-3p and miR-21-5p that are overex-pressed in PC tissues are released in the bloodstream from the tumour tissues, direct evidence for this is still lacking On the contrary, Let-7a-5p is a tumour suppres-sive miRNA that is downregulated in PC tissues as com-pared to normal or BPH tissues [44, 45] Hence, the decrease in Let-7a-5p plasma level in patients with aggressive PC is unlikely to be due to the release from cancer tissue More plausibly, lower expression level or reduced release of Let-7a-5p is genetically associated
Fig 5 Circulating Let-7a-5p levels in PC patients with low and high Gleason score Scatter plots show the log2RQ values of Let-7a-5p tested in EVs and in whole plasma of patients with Gleason score ≥ 8 (PC GH) and Gleason score ≤6 (PC GL) The mean log2RQ values and standard deviation is shown above each scatter plot Area under the ROC curve (AUC), 95% confidence interval and p value for differentiating between PC patients with high and low Gleason score is shown below each graph